Page 113 - 2014 Printable Abstract Book
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other pro- inflammatory cytokines. This response, however, is not involved in promoting persistent
genomic instability in this epithelial cell model. Supported by NASA-NSCOR grant NNX11AC3OG
(PS1-18) A siRNA synthetic lethality screen in small cell lung cancer cells identifies potential targets for
improved clinical outcome and genes involved in the DNA damage response. Matthew Z. Madden, BA
and David S. Yu, MD PhD, Emory University School of Medicine, Atlanta, GA
Purpose/Objectives: Small cell lung cancer (SCLC) accounts for 10% to 15% of lung cancer cases and is a
particularly aggressive form of the disease. Over the past 30 years clinical outcomes in SCLC have
improved relatively little compared to other cancers. Etoposide with platinum is a common first-line
combination chemotherapy for SCLC with median survival of 9 to 12 months. Both drugs induce damage
to DNA and initiate the DNA damage response (DDR). To discover possible SCLC drug targets, biomarkers
to better inform SCLC treatment, and/or novel DDR genes, we conducted a siRNA screen in a human SCLC
cell line to identify genes that, when knocked down, induce cytotoxicity themselves or sensitivity or
resistance to cisplatin and/or etoposide. Methodology: The siRNA screen was conducted in triplicate with
a library of “smartpool” siRNAs targeting 1,006 nuclear enzymes. NCI-H128 cells were transfected with
siRNA in 96 well plates, treated or not treated with cisplatin or etoposide 48 hours later, and assayed for
live cell number (viability) using resazurin reagent after another 72 hours. Treated-to-nontreated viability
ratios were calculated for each siRNA and normalized to the nonspecific siRNA control. Stringent hit
criteria were established based on the magnitude of viability change, p-value, and strictly standardized
mean difference of log2-transformed and untransformed viability ratios. Results and Future Directions: Z-
factors calculated with the positive sensitizing control siATR were 0.56 and 0.53 for the cisplatin and
etoposide screens, indicating high quality of hit separation. Multiple controls and other known DDR genes
in the siRNA library, including all ERCC genes for cisplatin sensitivity, were significantly sensitizing. 44
genes were selected for validation with a secondary deconvoluted siRNA screen in multiple cell lines.
Validated genes will be studied further for a role in the DDR and for possible clinical applications to
improve SCLC patient care.
(PS1-19) Investigating the cellular mechanisms of low-dose pulsed radiotherapy. Diane Schoenherr, BS;
Sarah A. Krueger, PhD; Sandra Galoforo, MS; George D. Wilson, PhD; Brian Marples, PhD, William
Beaumont Hospital, Royal Oak, MI
Our previous studies have demonstrated that low-dose pulsed radiotherapy (PRT) is a more
effective treatment than standard continously-delivered radiotherapy (SRT) for MYCN-amplified
neuroblastoma xenografts. PRT consists of 10 doses of 0.2 Gy with each individual 0.2 Gy treatment
separated by 3 minutes. More recently, PET data generated from F18-FDG and F18-MISO non-invasive
tumor imaging, ELISA analysis of circulating cytokines from harvested blood, and tumor
immunohistochemistry have indicated that changes in tumor microenvironment may explain the efficacy
of PRT. The aim of this study was to investigate the cell death mechanisms and damage signaling pathways
associated with PRT irradiation, with a mechanistic emphasis on low-dose hyper-radiosensitivity (HRS) as
an additional component to the efficacy of PRT. Irradiations were performed using an Xstrahl RS225 160
kVp X-ray machine with cells maintained at 37°C. The radiosensitivity of SK-N-BE (2), SK-N-SH, MC-IXC,
and SH-SY5Y neuroblastoma cells was determined after PRT and SRT by in vitro clonogenic survival assays.
111 | P a g e
genomic instability in this epithelial cell model. Supported by NASA-NSCOR grant NNX11AC3OG
(PS1-18) A siRNA synthetic lethality screen in small cell lung cancer cells identifies potential targets for
improved clinical outcome and genes involved in the DNA damage response. Matthew Z. Madden, BA
and David S. Yu, MD PhD, Emory University School of Medicine, Atlanta, GA
Purpose/Objectives: Small cell lung cancer (SCLC) accounts for 10% to 15% of lung cancer cases and is a
particularly aggressive form of the disease. Over the past 30 years clinical outcomes in SCLC have
improved relatively little compared to other cancers. Etoposide with platinum is a common first-line
combination chemotherapy for SCLC with median survival of 9 to 12 months. Both drugs induce damage
to DNA and initiate the DNA damage response (DDR). To discover possible SCLC drug targets, biomarkers
to better inform SCLC treatment, and/or novel DDR genes, we conducted a siRNA screen in a human SCLC
cell line to identify genes that, when knocked down, induce cytotoxicity themselves or sensitivity or
resistance to cisplatin and/or etoposide. Methodology: The siRNA screen was conducted in triplicate with
a library of “smartpool” siRNAs targeting 1,006 nuclear enzymes. NCI-H128 cells were transfected with
siRNA in 96 well plates, treated or not treated with cisplatin or etoposide 48 hours later, and assayed for
live cell number (viability) using resazurin reagent after another 72 hours. Treated-to-nontreated viability
ratios were calculated for each siRNA and normalized to the nonspecific siRNA control. Stringent hit
criteria were established based on the magnitude of viability change, p-value, and strictly standardized
mean difference of log2-transformed and untransformed viability ratios. Results and Future Directions: Z-
factors calculated with the positive sensitizing control siATR were 0.56 and 0.53 for the cisplatin and
etoposide screens, indicating high quality of hit separation. Multiple controls and other known DDR genes
in the siRNA library, including all ERCC genes for cisplatin sensitivity, were significantly sensitizing. 44
genes were selected for validation with a secondary deconvoluted siRNA screen in multiple cell lines.
Validated genes will be studied further for a role in the DDR and for possible clinical applications to
improve SCLC patient care.
(PS1-19) Investigating the cellular mechanisms of low-dose pulsed radiotherapy. Diane Schoenherr, BS;
Sarah A. Krueger, PhD; Sandra Galoforo, MS; George D. Wilson, PhD; Brian Marples, PhD, William
Beaumont Hospital, Royal Oak, MI
Our previous studies have demonstrated that low-dose pulsed radiotherapy (PRT) is a more
effective treatment than standard continously-delivered radiotherapy (SRT) for MYCN-amplified
neuroblastoma xenografts. PRT consists of 10 doses of 0.2 Gy with each individual 0.2 Gy treatment
separated by 3 minutes. More recently, PET data generated from F18-FDG and F18-MISO non-invasive
tumor imaging, ELISA analysis of circulating cytokines from harvested blood, and tumor
immunohistochemistry have indicated that changes in tumor microenvironment may explain the efficacy
of PRT. The aim of this study was to investigate the cell death mechanisms and damage signaling pathways
associated with PRT irradiation, with a mechanistic emphasis on low-dose hyper-radiosensitivity (HRS) as
an additional component to the efficacy of PRT. Irradiations were performed using an Xstrahl RS225 160
kVp X-ray machine with cells maintained at 37°C. The radiosensitivity of SK-N-BE (2), SK-N-SH, MC-IXC,
and SH-SY5Y neuroblastoma cells was determined after PRT and SRT by in vitro clonogenic survival assays.
111 | P a g e
